Process for the preparation of 1,2-dioxetane compounds and novel sulfur-substituted 1,2-dioxetane compounds as intermediates

ABSTRACT

A process for producing a stable triggerable dioxetane comprising; 
     (a) reacting a vinyl sulfide compound containing a sulfur-substituent SR 4 , wherein R 4  is an organic group containing 1 to 20 carbon atoms and optionally heteroatoms, with oxygen and light in the presence of a photosensitizer to form an intermediate sulfur-substituted dioxetane compound; and 
     (b) reacting the sulfur-substituted dioxetane compound with an electrophilic compound E--Y and a hydroxylic compound R 5  OH selected from the group consisting of alcohols, phenols and carboxylic acids or their salts and containing an OR 5  group to replace the SR 4  group of the dioxetane with the OR 5  group.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 371 of PCT/US96/09248 filed Jun. 19, 1996 and acontinuation-in-part of applicant's U.S. application Ser. No. 08/492,717filed on Jun. 20, 1995 now ABN.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a novel process for the preparation ofstable chemiluminescent 1,2-dioxetane compounds which can be triggeredto generate light. Stable, triggerable dioxetanes prepared by thepresent process are preferably of the formula: ##STR1## The presentinvention also relates to novel sulfur-substituted alkenes (vinylsulfides) preferably of the formula: ##STR2## and stabletriggerable-sulfur-substituted 1,2-dioxetanes preferably of the formula:##STR3## a process for their preparation and a process for their use asintermediates for producing stable triggerable 1,2-dioxetanessubstituted on the dioxetane ring with alkoxy, alkenyloxy, alkynyloxy,aryloxy, aralkyloxy or acyloxy groups.

(2) Dscription of Related Art

a. Synthesis of Dioxetanes

The preparation of dioxetanes with alkoxy substituents by addition ofsinglet oxygen to a vinyl ether is well known in the art. Singlet oxygenis typically produced by irradiation of a photosensitizing dye in thepresence of oxygen but can also be generated by thermolysis oftriphenylphosphite ozonide. Other methods of preparing dioxetanes withalkoxy substituents from vinyl ethers include electron-transferoxidation with oxygen and triarylaminium cation radical salts (R. Curci,L. Lopez, L. Troisi, S. M. K. Rashid and A. P. Schaap, Tetrahedron Lett.28, 5319-22 (1987); L. Lopez, L. Troisi and G. Mele, Tetrahedron Lett.32, 117-20 (1991)), oxidation by Cr(VI),or Mo(VI) oxide diperoxides (R.Curci, L. Lopez, L. Troisi, S. M. K. Rashid and A. P. Schaap,Tetrahedron Lett. 29, 3145-8 (1988)) and oxidation with triethylsilylhydrotrioxide (G. H. Posner, K. S. Webb, W. M. Nelson, T. Kishimoto andH. H. Seliger, J. Org. Chem., 54, 3252-4 (1989)). A dioxetane wasproduced in low yield by reaction of a dioxene compound with oxygenwhich had been passed through an electric discharge, apparentlyproducing a small amount of singlet oxygen in addition to ozone (T.-S.Fang and W.-P. Mei, Tetrahedron Lett. 28, 329-21 (1987)).

All of these methods for the preparation of alkoxy-substituteddioxetanes require the preparation of the precursor vinyl ether. Noreaction involving the direct introduction of alkoxy or aryloxy groupson a pre-formed dioxetane ring has been reported to the best ofapplicant's knowledge. There is thus a need for a general method for thepreparation of a variety of alkoxy-substituted dioxetanes from a commonintermediate which does not require the preparation of each individualvinyl ether precursor.

b. Sulfur-Substituted Dioxetanes

1,2-Dioxetanes with one or more sulfur-containing substituents on thedioxetane ring are known. All known examples are unstable, with mostdecomposing rapidly at room temperature. (W. Adam, L. A. Arias, D.Scheutzow, Tetrahedron Lett., 23(28), 2835-6 (1982); W. Adam, L. A.Encarnacion, Chem. Ber., 115(7), 2592-605 (1982); W. Ando, K. Watanabe,T. Migita, J. Chem. Soc., Chem. Commun. (24), 961-2 (1975); G. Geller,C. S. Foote, D. B. Pechmann, Tetrahedron Lett. 673-6 (1983); R. S.Handley, A. J. Stern, A. P. Schaap, Tetrahedron Lett. 26, 3183-6(1985)). The most stable sulfur-substituted dioxetanes, derived from4,5-dialkyl-2,3-dihydrothiophene decompose with a half-life of a fewminutes at room temperature (W. Adam, A. Griesbeck, K. Gollnick, K.Knutzen-Mies, J. Org. Chem., 53, 1492-5 (1988); K. Gollnick, K.Knutzen-Mies, J. Org. Chem., 56, 4027-31 (1991)). Twospiroadamantyl-substituted dioxetanes bearing one and two sulfursubstituents, respectively, on the dioxetane ring are known. Both havebeen reported to rapidly and completely decompose on attempted isolationat room temperature (W. Adam, L. A. Encarnacion, Chem. Ber., 115(7),2592-605 (1982)).

c. Synthesis of Vinyl Sulfides

Vinyl sulfides containing a carbon--carbon double bond and a sulfursubstituent directly attached to one of the double bond carbon atoms canbe prepared by various methods known to the skilled synthetic chemist.One of the classical methods for preparation of vinyl sulfides involvesthe reaction of a ketone and mercaptan with TiCl₄ and an amine base (T.Mukaiyama, K. Saigo, Chem. Lett., 479-82, 1973)). Vinyl sulfides havebeen formed by other methods which utilize titanium reagents. Vinylsulfones are reduced at the sulfur to vinyl sulfides with LiAlH₄ --TiCl₄(E. Akgun, K. Mahmood, C. A. Mathis, J. Chem. Soc., Chem. Commun, (6),761-2 (1994)). α-Halo-sulfoxides undergo elimination and reduction withZn--TiCl₄ to form vinyl sulfides (V. Retrakul, P. Poochaivatananon,Tetrahedron Lett., 24(5), 531-4 (1983)). In each of these methods, oneor both of the C--C bonds is already formed in the starting material.None of the foregoing methods involves the direct formation of thesulfur-substituted carbon--carbon double bond from two separate carbonatoms. No method of which Applicants are aware is known for creating avinyl sulfide by coupling two carbonyl-containing compounds, one ofwhich is a thioester, to form a double bond with a sulfur-substituent.

d. Chemically Triggerable Dioxetanes

Chemically triggerable adamantyl-stabilized dioxetanes are disclosed inU.S. Pat. No. 4,857,652 and a paper (A. P. Schaap, T. S. Chen, R. S.Handley, R. DeSilva, and B. P. Giri, Tetrahedron Lett., 1155 (1987)).These dioxetanes exhibit thermal half-lives of years but can betriggered to produce efficient chemiluminescence on demand. Benzofuranyldioxetanes substituted with trialkylsilyl and acetyl-protected phenolicgroups which produce weak chemiluminescence have also been reported (W.Adam, R. Fell, M. H. Schulz, Tetrahedron, 49(11), 2227-38 (1993); W.Adam, M. H. Schulz, Chem. Ber., 125, 2455-61 (1992)). Each of thesedioxetanes was prepared by dye-sensitized photooxygenation of a vinylether (alkene) precursor.

e. Enzymatically Triggerable Dioxetanes

Enzymatic triggering of adamantyl-stabilized 1,2-dioxetanes aredescribed in U.S. Pat. No. 4,857,652 and a series of papers (A. P.Schaap, R. S. Handley, and B. P. Giri, Tetrahedron Lett., 935 (1987); A.P. Schaap, M. D. Sandison, and R. S. Handley, Tetrahedron Lett., 1159(1987) and A. P. Schaap, Photochem. Photobiol., 47S, 50S (1988)). Thesedioxetanes bear a protected aryloxide substituent which is triggered todecompose with emission of light by the action of an enzyme in analkaline aqueous buffer to give an aryloxide intermediate dioxetanewhich decomposes with emission of light at a greatly increased rate.Chemiluminescence is thereby emitted at a much greater intensity thanthat resulting from slow thermal decomposition of the protected form ofthe dioxetane. Further examples of enzymatically triggered dioxetanesare disclosed in U.S. Pat. No. 5,068,339 to Schaap, U.S. Pat. Nos.5,112,960 and 5,220,005 and a PCT application (88 00695) to BronsteinU.S. Pat. No. 4,952,707 to Edwards, U.S. Pat. No. 5,132,204 to Urdea,U.S. Pat. No. 5,248,618 to Haces and PCT application WO94/10258 to Wangand in a publication (M. Ryan, J. C. Huang, O. H. Griffith, J. F. Keana,J. J. Volwerk, Anal. Biochem., 214(2), 548-56 (1993)). The enzymaticallytriggerable dioxetanes are now undergoing widespread use as substratesfor marker enzymes in numerous applications including immunoassays, geneexpression studies, Western blotting, Southern blotting, DNA sequencingand the identification of nucleic acid segments in infectious agents.

New processes for the preparation of existing and new triggerabledioxetanes are desirable to advance the state of the art. Processeswhich permit the preparation of dioxetane compounds which are difficultor impossible to prepare by known methods would be particularlydesirable. The process of the present invention provides such means.

OBJECTS

It is an object of the present invention to provide novel arylgroup-substituted 1,2-dioxetanes further substituted on the dioxetanering with a thioalkyl or thioaryl group SR₄. It is an object of thepresent invention to provide novel aryl group-substituted vinyl sulfidesand a process for their preparation, said vinyl sulfides being used inpreparing the sulfur-substituted dioxetanes. It is a further object ofthe present invention to provide a process for producing stable1,2-dioxetanes by replacement of the sulfur-containing group SR₄ of asulfur-substituted dioxetane with an alkoxy, alkenyloxy, alkynyloxy,aryloxy, aralkyloxy or acyloxy group OR₅. It is also an object of thepresent invention to provide a process of preparation of stable1,2-dioxetanes containing an alkoxy, alkenyloxy, alkynyloxy, aryloxy,aralkyloxy or acyloxy group OR₅ and further substituted on the dioxetanering with aryl group substituted with an OX substituent which can betriggered by activating agents to remove a protecting group X andconsequently generate chemiluminescence. It is a further object of thepresent invention to provide a general process for producing a number ofdifferent stable 1,2-dioxetanes each substituted on the dioxetane ringwith different alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy oracyloxy groups OR₅ by replacement of the sulfur-containing group SR₄ ofa sulfur-substituted dioxetane.

Dioxetane compounds prepared by the process of the present invention canbe used in assay methods to signal the presence or amount of an analyte,in emergency and novelty lighting applications and as light standardsfor luminometer calibration. Dioxetane compounds which are triggered byan activating agent to produce light are useful in immunoassays and thedetection of nucleic acids, antibodies, haptens and antigens bygenerally known methods.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a general process for the preparationof stable aryl group-substituted 1,2-dioxetanes (III) furthersubstituted on the dioxetane ring with an alkoxy, aryloxy or acyloxygroup which dioxetane can be triggered to generate chemiluminescence.The process involves the substitution of an alkoxy, alkenyloxy,alkynyloxy, aryloxy, aralkyloxy or acyloxy group OR₅ for a thioalkyl orthioaryl group SR₄ on the dioxetane ring of a sulfur-substituteddioxetane (II) mediated by an electrophilic compound E--Y. UntilApplicant's process was discovered, no process existed which wasgenerally applicable to the preparation of a series of stable dioxetanesfrom a common intermediate; each dioxetane was prepared by addition ofoxygen to the independently prepared alkene precursor. The success ofthe present process resides in the unanticipated discovery that certainsulfur-substituted dioxetanes can react with hydroxyl group-containingcompounds in the presence of an electrophile to replace the sulfursubstituent with an alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxyor acyloxy group OR₅.

Sulfur-substituted dioxetanes of the present invention are prepared byaddition of oxygen to a vinyl sulfide (I). The sequence of reactions isshown in the Scheme below. ##STR4## While not wishing to be bound by anyparticular theory, a plausible explanation for the reaction isillustrated in the Scheme shown below. ##STR5## No reaction involvingthe direct substitution of groups on a dioxetane ring has ever beenreported. The present invention represents the first demonstration ofthe synthesis of a dioxetane by replacing one of the dioxetane ringsubstituents of a different precursor dioxetane. The ability toselectively replace one of the ring substituents readily allows thesynthesis of a variety of new dioxetane compounds from a commonintermediate.

Sulfur-substituted dioxetanes useful in practicing the process of thepresent invention can be of the formula: ##STR6## wherein R₁ and R₂ areorganic groups providing sufficient stability to the dioxetane to permitconversion to an alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy oracyloxy-substituted dioxetane. The groups R₁ and R₂ are chosen frombranched chain or cyclic alkyl, substituted alkyl or heteroalkyl groups.R₁ and R₂ can optionally be joined together to form a cyclic orpolycyclic group which is spiro-fused to the dioxetane ring. The groupR₄ is selected from (C₁ -C₂₀) alkyl, (C₇ -C₃₀) aralkyl and (C₆ -C₃₀)aryl groups which can optionally contain non-interfering substituentsand optionally contain N, O, S, P or halogen heteroatoms within thealkyl, aralkyl or aryl group. The group R₃ is selected from aryl,biaryl, heteroaryl, fused ring polycyclic aryl or heteroaryl groups inwhich one or more of the ring hydrogens can be replaced by an atom orgroup selected from halogens, alkyl, alkoxy, substituted alkoxy,carbonyl, carboxyl, amino and alkylamino groups. The X group can be anyprotecting group which serves to block formation of the aryloxide anionand which can be replaced or removed as desired by an activating agentto form the aryloxide anion. Representative OX groups include hydroxyl,alkoxy, substituted alkoxy (e.g. methoxyethoxymethoxy (MEM-O) andtrimethylsilylethoxymethoxy (SEM-O)), acyloxy having the formula OOCR₁₀wherein R₁₀ is selected from alkyl and aryl groups containing 2 to 20carbon atoms, trialkylsilyloxy, triarylsilyloxy, aryldialkylsilyloxy,OPO(OR₈)₂ wherein R₈ is an organic group, oxygen pyranoside including,without limitation, β-D-galactosyloxy and β-D-glucuronidyloxy groups.

In a preferred embodiment, the process is used to form asulfur-substituted dioxetane (V) wherein the R₁ and R₂ groups are joinedtogether as a polycyclic alkyl group spiro-fused to the dioxetane ringas represented by the formula: ##STR7## wherein ##STR8## is thepolycyclic alkyl group. Most preferred is a sulfur-substituted dioxetanewherein the ##STR9## group is an adamantyl group with optionalnon-hydrogen substituents.

Electrophilic agents useful in practicing the present invention includebut are not limited to halogens including Cl₂, Br₂, I₂, ICl and IBr,hydrogen peroxide, singlet oxygen, pseudo-halogens such asN-chlorosuccinimide (NCS), N-bromosuccinimide (NBS) andN-iodosuccinimide (NIS), alkylating agents including alkyl halides andalkyl sulfates and sulfonates, transition metal salts, especially saltsof mercury, silver and gold, and Lewis acids such as titaniumtetrachloride. In one embodiment, the compound of structure E--Y mayserve as both the electrophilic agent and the hydroxylic compound R₅ O⁻M⁺. Compounds of this type described generically as (R₅ O⁻)_(n) M^(+n)wherein M is a metal with a strong propensity to react with sulfur, suchas silver, gold and especially, mercury, wherein n is 1, 2 or 3 andwherein R₅ O⁻ is an anion of a hydroxylic compound, in particular acarboxylate anion are effective to replace the SR₄ group of asulfur-substituted dioxetane with an OR₅ group. A preferred compound ofthis type is mercuric acetate Hg(OAc)₂.

Photosensitizers useful in practicing the process of the inventioninclude compounds known in the art to produce singlet oxygen uponirradiation with visible light in the presence of ground state oxygen.Exemplary photosensitizers include methylene blue, Rose Bengal, eosin,erythrosin, rhodamines, porphyrins, metal porphyrins and fullerenes.Photosensitizers may be employed as the soluble dye or linked to aninsoluble support such as silica or a polymer bead. Preferredphotosensitizers are methylene blue or polymer-bound Rose Bengal.

In a preferred mode of carrying out the inventive process, a vinylsulfide containing a sulfur substituent SR₄, wherein R₄ is an organicgroup containing 1 to 20 carbon atoms and optionally heteroatoms isconverted by low temperature photooxygenation to the correspondingsulfur-substituted 1,2-dioxetane by addition of singlet oxygen to thedouble bond. Progress of this reaction is readily monitored by thinlayer chromatography (TLC) or ¹ H NMR by observing the disappearance ofthe vinyl sulfide. Additionally, heating a small portion-of the reactionsolution leads to easily detectable chemiluminescence indicatingformation of the sulfur-substituted dioxetane. Visualization of thesulfur-substituted dioxetane by triggering with fluoride in DMSOproduces yellow-greenish chemiluminescence which is discernible to theunaided eye. Sulfur-substituted dioxetane compounds of relatively lowerthermal stability are preferably not isolated at this point but insteaddirectly reacted at a low temperature with a compound containing ahydroxyl group or a salt thereof. Sulfur-substituted dioxetane compoundsof relatively higher thermal stability can be first isolated beforereaction with a compound containing a hydroxyl group or a salt thereof.An electrophilic compound is added at low temperature to thesulfur-substituted dioxetane in an amount ranging from about 0.5 mol toabout 1.5 mol of electrophilic compound per mol of dioxetane based oncomplete conversion of the vinyl sulfide. It is especially preferredthat the photooxygenation and addition of electrophilic compound beperformed at or below about -40° C.; use of a Dry Ice-isopropyl alcoholmixture, ca. -78° C., for cooling is particularly suited for thispurpose. A hydroxylic compound R₅ --OH selected from alcohols, phenolswhere phenols are defined as aryl ring compounds with at least one OHsubstituent and carboxylic acids or their salts, if not already presentas the reaction solvent, is added at low temperature in an amount of atleast about 1 mol to 2 mol per mol of the vinyl sulfide. The hydroxyliccompound is permitted to react with the dioxetane to effect replacementof the SR₄ group with the OR₅ group.

Vinyl sulfide compounds (I) useful in practicing the present inventionare preferably of the formula: ##STR10## wherein R₁ and R₂ are organicgroups which can optionally be joined together to form cyclic orpolycyclic groups, wherein R₄ is selected from (C₁ -C₂₀) alkyl, (C₇-C₃₀) aralkyl and (C₆ -C₃₀) aryl groups and optionally containingheteroatoms, wherein R₃ is selected from aryl, biaryl, heteroaryl, fusedring polycyclic aryl or heteroaryl groups which can optionally containnon-interfering substituents and wherein X is a protecting group.Preferred R₁ and R₂ groups are selected from branched chain alkyl,cycloalkyl and aryl groups containing 3 to 20 carbon atoms andoptionally heteroatoms.

In a preferred embodiment, a vinyl sulfide having the formula: ##STR11##is used wherein ##STR12## is selected from cyclic and polycyclic organicgroups which can optionally be substituted. It is preferred that R₃ isan optionally substituted phenyl, naphthyl or other fused-ring arylgroup. It is especially preferred that R₃ is a phenyl group in which anOX group is oriented meta to the dioxetane ring group as shown below.The phenyl ring can optionally contain additional ring substituentsindependently selected from halogens, alkyl, substituted alkyl, alkoxy,substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups. Thegroup ##STR13## is more preferably a polycyclic group, preferably anadamantyl group optionally having one or more non-interferingsubstituent groups selected from halogens, alkyl, substituted alkyl,alkoxy, substituted alkoxy, carbonyl, carboxyl, phenyl, substitutedphenyl, amino and alkylamino groups covalently bonded thereto. Vinylsulfides useful in practicing the process of the present invention canbe prepared by art-known methods for the preparation of vinyl sulfides.An exemplary process for the preparation of vinyl sulfides is disclosedin T. Mukaiyama, K, Saigo, Chem. Lett., 479-82 (1973).

In another embodiment of the present invention, vinyl sulfide (IV) isformed by coupling a ketone compound and a thioester compound with a lowvalent titanium reagent exemplified by the following reaction: ##STR14##This reaction process achieves the direct formation of bothcarbon--carbon bonds of the vinyl sulfide product. The low valenttitanium reagent, which is used in excess, is prepared by reacting atitanium salt, preferably TiCl₃ or TiCl₄ with a metallic reducing agentselected from lithium, sodium, potassium, zinc and zinc-copper alloys ora metal hydride including, without limitation, sodium hydride, potassiumhydride, aluminum hydride, lithium aluminum hydride or potassiumtriethylborohydride in the presence or absence of an amine base in anaprotic solvent, preferably tetrahydrofuran. The ratio of ketone tothioester is preferably from about 1:3 to about 3:1. The reactionbetween the ketone and thioester is conducted between about 0° C. andthe reflux temperature of the solvent, preferably between about 20° C.and about 70° C.

Thioalkyl- and thioaryl-substituted dioxetanes of the present inventionare surprisingly stable and can be manipulated at temperatures of -10 to25° C. Reaction of the thioalkyl- and thioaryl-substituted dioxetanes ofthe present invention with an activating agent to cleave the O--X bondand remove the X group yields an unstable oxide intermediate dioxetanecompound is formed which decomposes and releases electronic energy toform light and two carbonyl-containing compounds of the formula:##STR15## In contrast, all dioxetanes bearing sulfur substituents knownin the prior art including those with an adamantyl substituent,spontaneously decompose at or, in most cases, well below roomtemperature. It was particularly unexpected that a sulfur-substituteddioxetane would exhibit decay rates in alkaline amine buffers containingmetal salts comparable to analogous alkoxy-substituted dioxetanes.

Stable alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy oracyloxy-substituted dioxetanes which can be prepared by the process ofthe present invention are of the formula: ##STR16## wherein R₁ and R₂are organic groups which provide stability and which can optionally bejoined together to form a cyclic or polycyclic group which isspiro-fused to the dioxetane ring, wherein R₅ is selected independentlyfrom the group consisting of (C₁ -C₂₀) alkyl groups, (C₂ -C₂₀) alkenylgroups, (C₂ -C₂₀) alkynyl groups, (C₆ -C₃₀) aryl groups, (C₇ -C₃₀)aralkyl groups and (C₁ -C₂₀) acyl groups. R₅ can optionally besubstituted with one or more substituents including, without limitation,hydroxy, alkoxy, halogen, cyano, nitro, amino, imine, ketone, aldehyde,carboxylic acid, carboxylic ester, carboxamide, thiol, thioester,trialkylsilyloxy, triarylsilyloxy and alkyldiarylsilyloxy substituents.The group R₃ is selected from aryl, biaryl, heteroaryl, fused ringpolycyclic aryl and fused ring polycyclic heteroaryl groups which canoptionally be substituted with non-interfering groups. X is a groupwhich can be removed by an activating agent as is generally known in theart to form an unstable oxide intermediate dioxetane compound. Inanother embodiment, X can be a group such as a hydrogen atom or a labilegroup which can be replaced with another removable group withouttriggering the decomposition of the dioxetane. Representative OX groupsinclude hydroxyl, alkoxy, substituted alkoxy (e.g. methoxyethoxymethoxy(MEM-O) and trimethylsilylethoxymethoxy (SEM-O)), acyloxy having theformula OOCR₁₀ wherein R₁₀ is selected from alkyl and aryl groupscontaining 2 to 20 carbon atoms, trialkylsilyloxy, triarylsilyloxy,aryldialkylsilyloxy, OPO(OR₈)₂ wherein R₈ is an organic group, OPO₃ ²⁻salts and oxygen pyranoside including, without limitation.β-D-galactosyloxy and β-D-glucuronidyloxy groups.

When chemiluminescence is to be generated the stable dioxetane istriggered with an activating agent to generate an unstable oxideintermediate dioxetane which decomposes and releases electronic energyto form light and two carbonyl containing compounds as shown in Scheme 4below. ##STR17##

A preferred dioxetane compound which can be made by the process of thepresent invention is a stable dioxetane of the formula: ##STR18##wherein ##STR19## is selected from cyclic and polycyclic organic groups,which can optionally be substituted with non-interfering groups andwhich is spiro-fused to the dioxetane ring and which provides thermalstability, wherein R₃, R₅ and X are as defined above so that when thedioxetane is triggered to remove the X group by an activating agent anunstable oxide intermediate dioxetane compound is formed whichdecomposes and releases electronic energy to form light and twocarbonyl-containing compounds of the formula: ##STR20## In a preferredembodiment, the group ##STR21## is a polycyclic organic groupspiro-fused to the dioxetane ring, containing 6 to 30 carbon atoms andwhich can optionally be substituted with non-interfering groups andwhich provides thermal stability. The group ##STR22## is more preferablyan adamantyl group optionally having at least one substituent groupselected from halogens, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, carbonyl, carboxyl, phenyl, substituted phenyl, amino andalkylamino groups covalently bonded thereto. In another preferredembodiment the group R₃ is a phenyl or naphthyl group. It is especiallypreferred that R₃ is a phenyl group in which the OX group as definedabove is oriented meta to the dioxetane ring group as shown below. Thephenyl ring can optionally contain additional ring substituentsindependently selected from halogens, alkyl, substituted alkyl, alkoxy,substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.##STR23##

The stable 1,2-dioxetane compounds have long half-lives at roomtemperature, typically ≦1 year, but can be triggered by an activatingagent to decompose rapidly with half-lives ranging from seconds to a fewminutes depending on the microenvironment where the dioxetane islocated.

Aryloxy-substituted dioxetanes of the formula: ##STR24## have beenprepared, it is believed for the first time, by the process of thepresent invention by using a phenol as the hydroxylic compound R₅ OH.Dioxetanes wherein Ar is an optionally substituted (C₆ -C₃₀) aryl groupand wherein X is a removable group are highly-stable compounds whichproduce visible light when triggered to remove X.

It is contemplated that the hydroxylic compound R₅ OH may itself beanother dioxetane molecule with a phenolic group. In this embodiment,chains of directly linked, oligomeric or polymeric dioxetanes areformed. Further, such oligomeric or polymeric dioxetanes can serve asthe hydroxylic compound in order to build chain structures of greaterlength. A requirement for this process is that the OX group of themonomeric, oligomeric or polymeric dioxetane serving as the R'₅ OHcomponent be present as OH. An example showing formation of a dimericbis-dioxetane (VII) is illustrated below to aid in understanding theprocess. ##STR25##

It will be obvious in view of the foregoing description that, underappropriate conditions, the intermediate carbonium ion can serve as itsown R₅ OH component; i.e. when X is H a self-condensation can occur toproduce an oligomeric or polymeric dioxetane chain compound. Asub-stoichiometric amount of a competing R'₅ OH compound is required toeffect chain termination. A possible dioxetane compound (VIII) preparedby this process is illustrated to aid in understanding the process.##STR26##

An acyloxy-substituted dioxetane of the formula: ##STR27## has beenprepared for the first time by the present process. In preparing adioxetane of this structure, the hydroxylic compound can be a carboxylicacid R₉ COOH, a salt thereof or it can be as the electrophilic compoundin the manner described above. Dioxetanes wherein R₉ is an optionallysubstituted (C₁ -C₂₀) alkyl or aryl group and wherein X is a removablegroup are also stable compounds which emit light when triggered toremove X.

A significant advantage of the present process for preparing alkoxy,alkenyloxy, alkynyloxy, aryloxy, aralkyloxy or acyloxy-substituteddioxetanes is that a single vinyl sulfide precursor compound can beconverted into any of a large number of other dioxetane compoundslimited only by the availability of the hydroxylic compound R₅ --OH orsalt R₅ O⁻ M⁺. Since the OR₅ group is not introduced until afterformation of the dioxetane ring, it is possible to prepare dioxetanecompounds which would be problematic to prepare by the prior art processof photooxygenating a vinyl ether precursor. The present process isamenable to the preparation of dioxetanes with groups which couldinterfere with the photooxygenation either by quenching of singletoxygen, or by quenching or bleaching the photosensitizer. The presentprocess is also amenable to the preparation of dioxetanes with groupswhich make the vinyl ether more electron-deficient and therefore lessreactive or unreactive to singlet oxygen. The present process furtherpermits the preparation of dioxetanes with groups such as C--C doublebonds which are themselves reactive toward singlet oxygen.

Dioxetanes prepared by the process of the present invention can be usedin a process for generating light which comprises reacting an activatingagent with a stable 1,2-dioxetane of the formula (III): ##STR28##wherein the X group is removed by the activating agent to form anunstable oxide intermediate dioxetane compound which decomposes andreleases electronic energy to form light and two carbonyl-containingcompounds of the formula: ##STR29##

In another embodiment, the activating agent can be a chemical, includingan enzyme, which reacts catalytically or stoichiometrically to triggerthe dioxetane. Exemplary activating agents are disclosed, for example,in U.S. Pat. No. 4,857,652 the relevant portion of the disclosure ofwhich is incorporated herein by reference. Activating agents which reactcatalytically or stoichiometrically to trigger the dioxetane by removalof the X group are well known in the art. Reactions can be conducted inaqueous solution, in which case it is often desirable to use one or moreart-known chemiluminescence enhancers to increase or prolong lightemission. Reactions can also be conducted in polar, aprotic solventssuch as dimethyl sulfoxide, N,N-dimethylformamide or acetonitrile.

The process for generating light can be performed in solution or on thesurface of a solid support such as a membrane. Dioxetanes prepared bythe process of the present invention can further be incorporated into achemiluminescent composition which comprises a dioxetane, one or moreenhancer substances and optional fluorescers. Enhancers are substanceswhich increase the amount of light produced on triggering the dioxetane.Enhancer substances well known in the art increase chemiluminescenceeither by providing a hydrophobic environment in which the lightemitting reaction can occur or through energy transfer to a fluorescentcompound held in proximity to the dioxetane.

The development of particular assays and kits using dioxetanes preparedby the process of the present invention will be readily apparent to theskilled artisan. The methods of use of the dioxetanes which can beprepared by the present process are illustrative of their utility only.The uses do not constitute a part of the invention per se.

EXAMPLES

In order to more fully describe the various aspects of the presentinvention, the following examples are described. The examples are to beconsidered illustrative and do not limit the scope of the invention.

Sulfur-substituted dioxetane compounds prepared using the process of thepresent invention are shown below.

                  TABLE 1    ______________________________________    Dioxetane Compounds    1 #STR30##    Dioxetane     R.sub.4      OX    ______________________________________    1             CH.sub.2 CH.sub.3                               OH    2             CH.sub.2 CH.sub.3                               OOCC(CH.sub.3).sub.3    3             4-C.sub.6 H.sub.4 F                               OH    4             4-C.sub.6 H.sub.4 F                               OOCC(CH.sub.3).sub.3    5             CH.sub.2 CF.sub.3                               OH    ______________________________________

Example 1 Synthesis of Compound 1

4-Ethylthio-4-(3-hydroxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1.sup.3,7 !decane!.

(a) Synthesis of 3-hydroxyphenyl tricyclo 3.3.1.1³,7 !-dec-2-yl ketone.To a stirred solution of 20.00 g (0.07397 mol) of(3-hydroxyphenyl)methoxymethylene!tricyclo 3.3.1.1³,7 !-decane(preparation described in U.S. Pat. No. 4,857,652) in 300 mL of methanolwas added 5 mL of concentrated hydrochloric acid. After about fifteenminutes, a white precipitate began to form. The reaction was allowed tostir overnight. Water (500 mL) was added to the reaction mixture, andthe precipitate was collected by suction filtration and washed with anadditional 500 mL of water. After air drying for a few hours, the solidwas dissolved in ethyl acetate and dried over magnesium sulfate. Themagnesium sulfate was-filtered and the ethyl acetate removed in vacuoyielding 23.42 g (97% yield) of the pure ketone as a white solid. ¹ HNMR (CDCl₃) δ1.55-2.05 (m, 12H), 2.309 (br s, 2H), 3.41 (br s, 1H),6.99-7.39 (m, 4H).

(b) Synthesis of (3-hydroxyphenyl)ethylthiomethylene!tricyclo 3.3.1.1³,7!decane. The ketone was converted to the vinyl sulfide by the method ofMukaiyama (T. Mukaiyama, K, Saigo, Chem. Lett., 479-82, 1973)). Anargon-purged round bottom flask was charged with 10.0 g (39 mmol) of thehydroxy ketone, 250 mL of anhydrous tetrahydrofuran (THF), and 9.5 mL(2.2 eq.) of TiCl₄. The resulting brown solution was stirred under argonfor 30 minutes at room temperature. A solution of 24.0 mL (4.4 eq.) oftriethylamine (Et₃ N) dried over KOH and 3.2 mL (1.1 eq.) of ethanethiolin 200 mL of dry THF was then added dropwise to the reaction solutionover a 2 hour period while the reaction solution darkened to a deepreddish brown. Stirring was maintained overnight. A portion of dry Et₃ N(100 mL) was added to the reaction solution causing the brief formationof some precipitate. The excess TiCl₄ was neutralized by the addition ofmethanol. This caused the formation of precipitate, turned the reactionmixture from brown to yellow to green, and caused the reaction mixtureto heat up. The reaction mixture was found to still be basic by pH paperand was allowed to stir for an hour. The reaction mixture had turnedback to a bright yellow color by the end of this hour. The mixture wasthen suction filtered and the white precipitate obtained was washed withethyl acetate and discarded. The solvents were then removed in vacuofrom the filtrate yielding a brownish yellow solid. This solid was thentaken up into 500 mL of ethyl acetate and the resulting mixture washedseveral times with water (emulsion formed). The ethyl acetate layer wasthen dried over magnesium sulfate, filtered and concentrated in vacuoyielding a viscous yellow oil which smelled of thiol. The oil waschromatographed on silica gel using methylene chloride as eluent. Thedesired vinyl sulfide and the starting ketone were recovered. Thesolvents were removed in vacuo from the first eluted fraction yielding8.57 g of light yellow oil which gradually solidified over the course ofseveral days. ¹ H NMR (CDCl₃) δ1.105 (t, 3H, J=7.3 Hz), 1.64-2.00 (m,12H), 2.238 (q, 2H, J=7.3 Hz), 2.64 (br s, 1H), 3.58 (br s, 1H), 4.813(s, 1H), 6.69-6.83 (m, 3H), 7.175 (t, 1H).

(c) Synthesis of 4-ethylthio-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1.sup.3,7 !decane! (1). A smallphotooxygenation apparatus was charged with 102 mg of the vinyl sulfide,1-2 mg of methylene blue, and 10 mL of methylene chloride dried overmagnesium sulfate. The resulting solution was then cooled to -78° C.with oxygen bubbling through it. After several minutes, the reactionsolution was irradiated with a 1000 W sodium lamp (GE LUCALOX) shieldedby a 0.005" polyimide film (DuPont) for 20 min to produce the dioxetane.Mixing a sample of the dioxetane with an excess of tetrabutylammoniumfluoride (TBAF) in DMSO produced yellow chemiluminescence.

Example 2 Synthesis of Compound 2

4-Ethylthio-4-(3-pivaloyloxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo-3.3.1.1³,7 !decane!.

(a) Synthesis of 3-pivaloyloxyphenyl-tricyclo 3.3.1.1³,7 !-dec-2-ylketone. 3-Hydroxyphenyl-tricyclo 3.3.1.1³,7 !dec-2-yl ketone (2.1 g, 8.2mmol) was dissolved in 200 mL of CH₂ Cl₂ and 4.6 mL of Et₃ N (4 eq.).Pivaloyl chloride (1.1 mL, 1.1 eq.) was added dropwise to the solutionand the reaction stirred for two hours to complete acylation of thephenol group. The solution was extracted with three 100 mL portions ofwater and dried over MgSO₄. Evaporation of the solvent left a colorlessoil which slowly solidified. Yield 2.77 g; ¹ H NMR (CDCl₃) δ1.369 (s,9H), 1.55-2.05 (m, 12H), 2.307 (br s, 2H), 3.422 (br s, 1H), 7.20-7.68(m, 4H).

(b) Synthesis of (3-pivaloyloxyphenyl)ethylthiomethylene!-tricyclo3.3.1.1³,7 !decane. An Ar-purged round bottom flask was charged with0.50 g (1.47 mmol) of the pivaloyloxy ketone, 20 mL of dry THF, and 0.16mL (1 eq.) of TiCl₄. The resulting orange solution was treated with asolution of 0.43 mL (2.1 eq.) of dry Et₃ N and 0.12 mL (1.1 eq.) ofethanethiol in 20 mL of dry THF added dropwise to the reaction solutionover a 2 hour period. The reaction solution darkened to a deep reddishbrown. Stirring was maintained for about 65 hours. The excess TiCl₄ wasneutralized by the addition of 5 mL of methanol. The solvents were thenremoved in vacuo yielding a yellow oil. The oil was chromatographed onsilica gel using 5-20% ethyl acetate in hexane as eluent yielding 0.21 gof a colorless oil: ¹ H NMR (CDCl₃) δ1.096 (t, 3H, J=7.2 Hz), 1.355 (s,9H), 1.68-1.97 (m, 12H), 2.238 (q, 2H, J=7.2 Hz), 2.66 (br s, 1H), 3.59(br s, 1H), 6.91-7.32 (m, 4H); ¹³ C NMR (CDCl₃) δ15.11, 25.89, 27.13,28.04, 30.29, 35.48, 35.57, 36.96, 39.03, 39.36, 119.59, 120.41, 122.50,126.66, 128.57, 141.44, 150.91, 153.22, 176.86.

(c) Synthesis of 4-ethylthio-4-(3-pivaloyloxyphenyl)spiro-1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane! (2). A smallphotooxygenation apparatus was charged with 60 mg (0.16 mmol) of thevinyl sulfide, 180 mg of polymer-bound Rose Bengal (U.S. Pat. No.4,315,998), and 20 mL of CH₂ Cl₂ dried over MgSO₄. The resulting mixturewas then cooled to -78° C. with oxygen bubbling through it. Afterseveral minutes, the reaction solution was irradiated with a 1000 Wsodium lamp for 25 min. TLC using 10% ethyl acetate in hexane showedconversion to a new material which emitted light when heated. Thesensitizer was filtered off, the solvent evaporated at 0° C. and thematerial examined by ¹ H NMR. TLC suggested a higher degree ofdecomposition than was evident from the NMR spectrum suggesting that thedioxetane is less stable on silica. Mixing a sample of the dioxetanewith an excess of TBAF in DMSO produced yellow chemiluminescence. ¹ HNMR (CDCl₃) δ1.117 (t, 3H, J=7.2 Hz), 1.20-2.1 (m, 21H), 2.21 (br s,1H), 2.27-2.40 (m, 2H), 3.11 (br s, 1H), 7.04-7.60 (m, 4H).

Example 3 Synthesis of Compound 3

4-(p-Fluorophenylthio)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!.

(a) Synthesis of(3-hydroxyphenyl)-(p-fluorophenylthio)-methylene!tricyclo 3.3.1.1³,7!decane. A solution of 10.33 g (40.3 mmol) of the hydroxy ketone ofExample 1(a) and 250 mL of dry THF was purged with argon and treatedwith 9.8 mL (2.2 eq.) of TiCl₄. After 30 min, the resulting brownsolution was treated with a solution of 25.0 mL (4.4 eq.) of dry Et₃ Nand 4.8 mL (1.1 eq.) of 4-fluorothiophenol in 200 mL of dry THF addeddropwise to the reaction solution over a 2 hour period. The reactionsolution darkened to a deep reddish brown while stirred over night. DryEt₃ N (100 mL) was added, changing the color to yellow. The excess TiCl₄was neutralized by the addition of 2 mL of ethanol. The solvents werethen removed in vacuo from the filtrate yielding a brownish yellowsolid. This solid was then taken up into 500 mL of ethyl acetate and theresulting mixture washed several times with water (emulsion formed). Theethyl acetate layer was then dried over magnesium sulfate, filtered andconcentrated in vacuo yielding a viscous yellow oil which smelled of thethiophenol. The oil was chromatographed on silica gel using methylenechloride as eluent. The vinyl sulfide was collected in two fractions,the first being contaminated with the thiophenol, the second being pureproduct. The first fraction was chromatographed on silica gel using10-20% ethyl acetate in hexane as eluent yielding a second crop of vinylsulfide which combined with the previous crop yielded 11.7 g: ¹ H NMR(CDCl₃) δ1.75-2.05 (m, 12H), 2.783 (br s, 1H), 3.674 (br s, 1H), 4.559(s, 1H), 6.57-7.118 (m, 8H).

(b) Synthesis of 4-(p-Fluorophenylthio)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !-decane! (3). A smallphotooxygenation apparatus was charged with 54 mg of the vinyl sulfide,1-2 mg of methylene blue, 5 mL of CH₂ Cl₂ (dried over MgSO₄) and 5 mL ofCF₃ CH₂ OH. The resulting solution was then cooled to -40° C. withoxygen bubbling through it. After several minutes, the reaction solutionwas irradiated with a 1000 W sodium lamp for 24 min to produce thedioxetane as shown by the appearance of a new material by TLC whicheluted immediately below the alkene and emitted light when heated.

Example 4 Synthesis of Compound 4

4-(p-Fluorophenylthio)-4-(3-pivaloyloxyphenyl)spiro1,2-dioxetane-3,2'tricyclo- 3.3.1.1³,7 !decane!.

(a) Synthesis of(3-pivaloyloxyphenyl)-(p-fluorophenylthio)methylene!tricyclo3.3.1.1.sup.3,7 !decane. The pivalate-protected ketone described inexample 3(a) was converted to the vinyl sulfide by stirring 0.63 g (1.85mmol) of the ketone, 30 mL of dry THF and 0.22 mL (1.1 eq.) of TiCl₄.The resulting orange solution was stirred under argon for 5 minutes. Asolution of 0.57 mL (2.2 eq.) of Et₃ N and 0.22 mL (1.1 eq.) ofp-fluorothiophenol in 30 mL of dry THF was then added dropwise to thereaction solution over 30 min while the reaction solution turned purple.Stirring was maintained for ca. 72 hours. The excess TiCl₄ wasneutralized by the addition of ca. 2 mL of ethanol, the solventsevaporated and the residue purified by chromatography using 1-10% ethylacetate in hexane. The alkene (0.35 g) was obtained as an oil: ¹ H NMR(CDCL₃) δ1.330 (s, 9H), 1.70-2.05 (m, 12H), 2.775 (br s, 1H), 3.694 (brs, 1H), 6.78-7.20 (m, 8H).

(b) Synthesis of 4-(p-Fluorophenylthio)-4-(3-pivaloyloxyphenyl)spiro1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7 !decane! (4). A smallphotooxygenation apparatus was charged with 54 mg (0.12 mmol) of thevinyl sulfide, 150 mg of polymer-bound Rose Bengal, and 25 mL of CH₂ Cl₂dried over MgSO₄. The resulting solution was then cooled to -78° C. withoxygen bubbling through it. After several minutes, the reaction mixturewas irradiated with a 1000 W sodium lamp for 3.5 hours. TLC using 10%ethyl acetate in hexane showed conversion to a new material whichemitted light when the plate was heated. The sensitizer was filteredoff, the solvent evaporated and the material purified by chromatographyon silica with 20% ethyl acetate in hexane which yielded 30 mg ofslightly yellow oil which emitted light when a sample was heated on aTLC plate and additionally produced red chemiluminescence when triggeredwith DMSO/TBAF. ¹ H NMR (CDCl₃) δ1.375 (s, 9H), 1.68-1.97 (m, 12H), 2.66(br s, 1H), 3.59 (br s, 1H), 6.74-7.95 (m, 8H).

Example 5 Synthesis of Compound 5

4-(2,2,2-trifluoroethylthio)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!.

(a) Synthesis of (3-hydroxyphenyl)(2,2,2-trifluoroethylthio)methylene!tricyclo 3.3.1.1³,7 !decane. Anargon-purged round bottom flask was charged with 1.0 g (3.9 mmol) of theketone of Example 2(a), 40 mL of anhydrous THF and 1.0 mL (2.3 eq.) ofTiCl₄. The resulting brown solution was stirred under argon for 30minutes at room temperature. A solution of 2.4 mL (4.4 eq.) oftriethylamine dry Et₃ N and 0.38 mL (1.1 eq.) of2,2,2-trifluoroethanethiol in 60 mL of dry THF was then added dropwiseto the reaction solution over 1.5 hours while the reaction solutiondarkened to a deep reddish brown. Stirring was maintained overnight. Aportion of dry Et₃ N (10 mL) was added to the reaction solution followedby 10 mL of methanol. The reaction mixture was allowed to stir for anhour and then suction filtered. The filtrate was evaporated yielding agummy yellow solid. This solid was then taken up in CH₂ Cl₂ and theresulting mixture washed with water (emulsion formed). The CH₂ Cl₂ layerwas then dried over MgSO₄, filtered and concentrated in vacuo yielding ayellow solid which smelled of thiol. The solid was chromatographed onsilica gel using CH₂ Cl₂ as eluent. The vinyl sulfide and the startingketone were recovered. The solvents were removed in vacuo from the firsteluted fraction yielding 0.16 g of light yellow oil. ¹ H NMR (CDCl₃)δ1.65-2.02 (m, 12H), 2.624 (br s, 1H), 2.791 (q, 2H, J=9.8 Hz coupled toF), 3.609 (br s, 1H), 4.892 (s, 1H), 6.70-6.86 (m, 3H), 7.207 (t, 1H);¹³ C NMR (CDCl₃) δ27.949, 33.899 (q, J=31 Hz), 35.901, 35.962, 36.842,38.906, 39.240, 114.244, 116.611, 122.591, 125.70 (q, J=277 Hz),129.420, 140.226, 155.190, 155.403.

(b) Synthesis of 4-(2,2,2-trifluoroethylthio)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !-decane! (5). A smallphotooxygenation apparatus was charged with 47.2 mg of the vinylsulfide, 2 mg of methylene blue, and 20 mL of dry CH₂ Cl₂. The resultingsolution was then cooled to -78° C. with oxygen bubbling through it.After several minutes, the reaction solution was irradiated with a 1000W sodium lamp for 45 min to produce mainly the dioxetane as shown by thelight emitted on heating the TLC plate. The solution was concentratedand the product isolated by preparative TLC using 20% ethyl acetate inhexane yielding 30.4 mg of the product as a slightly yellow oil. Mixinga sample of the dioxetane with excess TBAF in DMSO producedorange-yellow light. ¹ H NMR (CDCl₃) δ1.16-2.10 (m, 12H), 2.31 (br s,1H), 2.948 (dq, 2H), 3.02 (br s, 1H), 5.35 (br s, 1H), 6.878 (dd, 1H),7.14-7.38 (m, 3H).

                  TABLE 2    ______________________________________    Alkoxy and Aryloxy Dioxetane Compounds    2 #STR31##    Dioxetane  R              X    ______________________________________     6         CH.sub.3       H     7         CH(CH.sub.3).sub.2                              H     8         CH.sub.2 CH═CH.sub.2                              H     9         CH.sub.2 CH.sub.2 CN                              H    10         CH.sub.2 CH.sub.2 OH                              H    11         C.sub.6 H.sub.5                              H    12         COCH.sub.3     H    13         CH.sub.2 CH.sub.3                              H    14         CH(CF.sub.3).sub.2                              H    15         CH.sub.2 CCl.sub.3                              H    16         CH.sub.2 CHCl.sub.2                              H    17         CH.sub.2 CF.sub.2 CF.sub.3                              H    18         CH.sub.2 CF.sub.2 CF.sub.2 CF.sub.3                              H    19         2,6-difluorophenyl                              H    20         CH.sub.3       COC(CH.sub.3).sub.3    21         CH.sub.3       Si(CH.sub.3).sub.2 t-Bu    22         CH.sub.3       SiPh.sub.2 t-Bu    23         CH.sub.3       PO(OCH.sub.2 CH.sub.2 CN).sub.2    24         CH.sub.3       PO.sub.3 Na.sub.2    ______________________________________

Example 6 Synthesis of Compound 6

4-(3-Hydroxyphenyl)-4-methoxyspiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1.sup.3,7 !decane!. The vinyl sulfide of Example 1(b) (0.113 g)was irradiated for 15 min in 20 mL of methanol containing a few crystals(<1 mg) of methylene blue with continuous oxygen bubbling at -78° C.using a 1000 W Na lamp. Iodine (0.096 g) and 1 mL of 30% H₂ O₂ wereadded and the mixture warmed to room temperature. Progress of thereaction was monitored by the disappearance of starting material by TLC(20% ethyl acetate in hexane) and the appearance of new bands whichemitted blue light when the plate was heated and by observing the colorof chemiluminescence from an aliquot of the reaction solution reactedwith 0.1 M TBAF in DMSO. The yellow emission of dioxetane 1 wasgradually replaced by the blue emission of dioxetane 6 over a period ofseveral hours. The solution was concentrated to 1-2 mL and separated onpreparative TLC with 10% ethyl acetate in hexane. The band containingdioxetane 6 was collected, desorbed and evaporated yielding 77 mg.

Example 7 Alternate Synthesis of Compound 6

The vinyl sulfide of Example 1(b) (0.104 g) was irradiated for 23 min in10 mL of CH₂ Cl₂ containing a few crystals (<1 mg) of methylene bluewith continuous oxygen bubbling at -78° C. using a 1000 W Na lamp.N-Chlorosuccinimide (42.8 mg) was added. After 10 min, 28 μL of methanol(2 eq.) was added and the solution warmed to room temperature. After anadditional 30 min, TLC showed a new material which emitted blue lightwhen the plate was heated. The solution was evaporated, the blue residuewashed with ether and the ether evaporated to produce an oil. This oilwas redissolved in a small amount of CH₂ Cl₂. The product was separatedby preparative TLC using 20% ethyl acetate in hexane yielding 50 mg ofthe product as a white solid.

Example 8 Synthesis of Compound 7

4-Isopropoxy-4-(3-hydroxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1.sup.3,7 !decane!. The vinyl sulfide of Example 1(b) (0.131 g)was irradiated for 13 min in 20 mL of 2-propanol containing a fewcrystals (<1 mg) of methylene blue with continuous oxygen bubbling at-78° C. using a 1000 W Na lamp. Iodine (0.111 g) and 1 mL of 30% H₂ O₂were added and the mixture warmed to room temperature. Progress of thereaction was monitored by the appearance of a new material by TLC (40%ethyl acetate in hexane) which emitted blue light when the plate washeated and by observing the color of chemiluminescence from an aliquotof the reaction solution reacted with 0.1 M TBAF in DMSO. The yellowemission of dioxetane 1 was gradually replaced by the blue emission ofdioxetane 7 over a period of several hours. The solution wasconcentrated to 1-2 mL and separated on preparative TLC with 10% ethylacetate in hexane. A band containing dioxetane 7 and adamantanone wascollected, desorbed and evaporated yielding 24 mg of a white solid(Dioxetane 7): ¹ H NMR (CDCl₃) δ1.160 (d), 1.257 (d), 1.22-2.14 (m),3.08 (br s), 3.747 (sept), 5.641 (s), 6.892 (dd), 7.2 (br s), 7.25-7.33(m).

A second component was also isolated as a pale yellow oil whichexhibited an NMR spectrum consistent with the structure: ##STR32## ¹ HNMR (CDCl₃) δ1.145 (d), 1.253 (d), 1.20-2.14 (m), 3.06 (br s), 3.715(sept), 5.77 (br s), 6.8-7.5 (m), 7.707 (d).

Example 9 Synthesis of Compound 8

4-(3-Hydroxyphenyl)-4-(2-propenyloxy) spiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1³,7 !decane!. The vinyl sulfide of Example 1(b) (0.100 g) wasirradiated in 20 mL of CH₂ Cl₂ containing a few crystals (<1 mg) ofmethylene blue with continuous oxygen bubbling at -78° C. using a 1000 WNa lamp. TLC indicated that the vinyl sulfide was consumed after 10 min.N-Chlorosuccinimide (0.0446 g) was added and the mixture maintained at-78° C. for 15 minutes. Allyl alcohol (50 uL) was added and the solutionwarmed to room temperature. Progress of the reaction was monitored bythe appearance of a new material by TLC (20% ethyl acetate in hexane)which emitted blue light when the plate was heated and by observing thecolor of chemiluminescence from an aliquot of the reaction solutionreacted with 0.1 M TBAF in DMSO. The yellow emission of dioxetane 1 wasgradually replaced by the blue emission of dioxetane 8. The solution wasconcentrated to dryness and the residue extracted with ether. The etherwas evaporated and the resulting residue chromatographed on a silicaprep. TLC plate with 20% ethyl acetate in hexane. The major bandcontained 47 mg of dioxetane 8: ¹ H NMR (CDCl₃) δ1.09 (m, 1H), 1.26 (m,1H), 1.471 (dq, 1H), 1.51-1.96 (m, 9H), 2.195 (br s, 1H), 3.127 (br s,1H), 3.72-3.82 (m, 1H), 4.02-4.12 (m, 1H), 5.14-5.22 (m, 1H), 5.32-5.44(m, 2H, vinylic H and phenolic-OH), 5.90-6.03 (m, 1H), 6.86-6.93 (m,1H), 7.19 (br s, 2H), 7.26-7.34 (m, 1H).

Example 10 Synthesis of Compound 9

4-(2-Cyanoethoxy)-4-(3-hydroxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo-3.3.1.1³,7 !decane!. The vinyl sulfide of Example 1(b) (0.052 g) wasirradiated in 10 mL of CH₂ Cl₂ containing a few crystals (<1 mg) ofmethylene blue with continuous oxygen bubbling at -78° C. using a 1000 WNa lamp for 10 min. N-Chlorosuccinimide (0.0229 g) was added and themixture maintained at -78° C. for 15 minutes. 2-Cyanoethanol (25.8 uL)in 3 mL of dry CH₂ Cl₂ was added and the solution warmed to roomtemperature. Progress of the reaction was monitored by the appearance-ofa new material by TLC (20% ethyl acetate in hexane) which emitted bluelight when the plate was heated and by observing the color ofchemiluminescence from an aliquot of the reaction solution reacted with0.1 M TBAF in DMSO. The yellow emission of dioxetane 1 was graduallyreplaced by the blue emission of dioxetane 9 over a period of 45 min.The solution was concentrated to dryness and the residue dissolved in aminimal amount of CH₂ CL₂ and purified by prep. TLC with 25% ethylacetate in CH₂ Cl₂ ; ¹ H NMR (CDCl₃) δ1.01-1.11 (m, 1H), 1.23-1.32 (M,1H), 1.45-1.94 (m, 10H), 2.23 (br s, 1H), 2.60-2.72 (m, 1H), 2.76-2.90(m, 1H), 3.06 (br s, 1H), 3.49-3.60 (m, 1H), 3.69-3.81 (m, 1H), 5.93 (brs, 1H), 6.88-6.98 (m), 7.19 (br s, 2H), 7.325 (t, 1H).

Example 11 Synthesis of Compound 10

4-(2-Hydroxyethoxy)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. The vinyl sulfide ofExample 1(b) (0.099 g) was irradiated for 11.5 min in 15 mL of CH₂ Cl₂containing a few crystals (<1 mg) of methylene blue with continuousoxygen bubbling at -78° C. using a 1000 W Na lamp. TLC indicated thatthe vinyl sulfide was consumed. N-Chlorosuccinimide (0.044 g) was addedand the mixture maintained at -78° C. for 15 minutes. Ethylene glycol(37 uL) was added and the solution warmed to room temperature. Progressof the reaction was monitored by the appearance of a new material by TLC(20% ethyl acetate in hexane) which emitted blue light when the platewas heated and by observing the color of chemiluminescence from analiquot of the reaction solution reacted with 0.1 M TBAF in DMSO. Theyellow emission of dioxetane 1 was gradually replaced by the blueemission of dioxetane 10 over a period of an hour. The solution wasconcentrated to dryness and the residue extracted with ether. The etherwas evaporated and the resulting residue chromatographed on a silicaprep. TLC plate with 40% ethyl acetate in hexane. The major bandcontained 18 mg of dioxetane 10.

Example 12 Synthesis of Compound 11

4-(3-Hydroxyphenyl)-4-phenoxyspiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1.sup.3,7 !decane!. The vinyl sulfide of Example 1(b) (0.106 g)was irradiated for 10 min in 20 mL of CH₂ Cl₂ containing a few crystals(<1 mg) of methylene blue with continuous oxygen bubbling at -78° C.using a 1000 W Na lamp. TLC indicated that the vinyl sulfide wasconsumed. N-Chlorosuccinimide (0.047 g) was added and the mixturemaintained at -78° C. for 15 minutes. Phenol (83 mg) was added and thesolution warmed to room temperature. Progress of the reaction wasmonitored by the appearance of a new material by TLC (20% ethyl acetatein hexane) which emitted blue light when the plate was heated and byobserving the color of chemiluminescence from an aliquot of the reactionsolution reacted with 0.1 M TBAF in DMSO. The yellow emission ofdioxetane 1 was gradually replaced by the blue emission of dioxetane 11over a period of an hour. The solution was concentrated and the residuechromatographed on a silica prep. TLC plate with 20% ethyl acetate inhexane. The major band was isolated and found to contain a mixture ofphenol, ca. 60 mg of dioxetane 11 and ca. 5 mg of adamantanone.Dioxetane 11: ¹ H NMR (CDCl₃) δ1.09-1.19 (m), 1.25-1.35 (m), 1.45-1.54(m), 1.56-2.14 (m), 2.26 (br s), 3.28 (br s), aromatic peaks could notbe resolved from residual phenol.

Example 13 Synthesis of Compound 12

4-Acetoxy-4-(3-hydroxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo3.3.1.1.sup.3,7 !decane!. The vinyl sulfide of Example 1(b) (50 mg) wasirradiated for 25 min in 20 mL of CH₂ Cl₂ containing 2 mg of methyleneblue with continuous oxygen bubbling at -78° C. using a 1000 W Na lamp.Mercuric acetate (400 mg) in 5 mL of 1,1,1,3,3,3-hexafluoro-2-propanolwas added. TLC indicated the formation of two new products which emittedlight upon heating the plate. The solution was evaporated, the blueresidue was extracted with ether, the ether solution filtered andevaporated to a blue oil. The oil was redissolved in a small amount ofCH₂ Cl₂ and subjected to preparative TLC using 20% ethyl acetate inhexane. A band was isolated which contained 40 mg of dioxetane 12. Asmall quantity of dioxetane 14 was also obtained. Dioxetane 12: ¹ H NMR(CDCl₃) δ1.06 (m, 1H), 1.27 (m, 1H), 1.48 (m, 1H), 1.54-1.90 (m, 9H),2.193 (s, 3H), 2.26 (br s, 1H), 3.06 (br s, 1H), 5.66 (br s, 1H), 6.80(m, 1H), 7.12 (br s, 2H), 7.248 (t, 1H).

Example 14 Synthesis of Compound 13

A small photooxygenation apparatus was charged with 99.1 mg of the vinylsulfide of Example 3(a), 1-2 mg of methylene blue, 10 mL of CH₂ Cl₂ and10 mL of absolute ethanol. The resulting solution was then cooled to 0°C. with oxygen bubbling through it. After several minutes, the reactionsolution was irradiated with a 1000 W sodium lamp for 15 min. NCS (40.0mg, 1.1 eq.) was added, the reaction maintained at 0° C. for 60 min andthen at room temperature for 3 h. The solvents were evaporated and theresidue purified by preparative TLC, eluting with CH₂ Cl₂. A colorlessoil (47.6 mg) was produced. Dioxetane 13: ¹ H NMR (CDCl₃) δ1.05-1.90 (m,15H), 2.17 (br s, 1H), 3.09 (br s, 1H), 3.29 (m, 1H), 3.55 (m, 1H), 5.68(br s, 1H), 6.86-7.36 (m, 4H).

Example 15 Synthesis of Compound 14

4-(Hexafluoroisopropoxy)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. The vinyl sulfide ofExample 2(b) (0.102 g) was irradiated for 10 min in 20 mL of CH₂ Cl₂containing 2 mg of methylene blue with continuous oxygen bubbling at-78° C. using a 1000 W Na lamp. The solution was poured into a solutionof 5 mL of 1,1,1,3,3,3-hexafluoro-2-propanol in 10 mL of CH₂ Cl₂.Mercuric acetate (ca. 50 mg) in 2 mL of hexafluoro-2-propanol was addedgradually and the solution stirred for 30 min. TLC showed two newmaterials which emitted light when the plate was heated. The solutionwas evaporated, the blue residue washed with ether and the etherevaporated to produce an oil. This oil was redissolved in a small amountof CH₂ Cl₂ and the product isolated by preparative TLC using 30% ethylacetate in hexane yielding 8.1 mg of the product as a colorless oil.Reaction of a sample of the dioxetane with an excess oftetrabutylammonium fluoride in DMSO produced green chemiluminescence. ¹H NMR (CDCl₃) 81.13 (m, 1H), 1.32 (m, 1H), 1.46-2.16 (m, 10H), 2.234 (brs, 1H), 2.994 (br s, 1H), 4.646 (br s, 1H), 5.326 (br s, 1H), 6.86-7.40(m, 4H); ¹⁹ F NMR (CDCl₃) δ(rel. to CFCl₃) -72.316, -72.013 (d).

Example 16 Synthesis of Compound 15

4-(3-Hydroxyphenyl)-4-(2 2,2-trichloroethoxy)spiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. The vinyl sulfide(3-hydroxyphenyl)-ethylthiomethylene!tricyclo 3.3.1.1³,7 !decane (102mg) was irradiated for 20 min in 10 mL of CH₂ Cl₂ containing 1 mg ofmethylene blue with continuous oxygen bubbling at -40° C. using a 1000 WNa lamp. N-Chlorosuccinimide (1 eq.) was added. After 10 min, 3 mL of2,2,2-trichloroethanol was added and the solution warmed to roomtemperature. After an additional 30 min, TLC showed a new material whichemitted blue-green light when the plate was heated. The solution wasevaporated, redissolved in a small amount of CH₃ OH and evaporated. Theproduct was separated by preparative TLC using 20% ethyl acetate inhexane yielding 53.5 mg of the product as a slightly yellow oil whichcontained some decomposition products: ¹ H NMR (CDCl₃) 81.12 (m, 1H),1.30 (m, 1H), 1.46-2.16 (m, 10H), 2.227 (br s, 1H), 3.198 (br s, 1H),3.909 (d, 1H, J=10.3 Hz), 4.133 (d, 1H, J=10.3 Hz) 5.75 (br s, 1H),6.940 (dd, 1H), 7.06-7.42 (m, 3H).

Example 17 Synthesis of Compound 16

4-(2,2-Dichloroethoxy)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. The vinyl sulfide(3-hydroxyphenyl)ethylthiomethylene!tricyclo 3.3.1.1³,7 !decane (111 mg)was irradiated for 16 min in 10 mL of CH₂ Cl₂ containing 1 mg ofmethylene blue with continuous oxygen bubbling at -78° C. using a 1000 WNa lamp. N-Chlorosuccinimide (50 mg, 1 eq.) was added. After 10 min,60.7 μL of 2,2-dichloroethanol (2 eq.) was added and the solution warmedto room temperature. After an additional 30 min, TLC showed a newmaterial which emitted blue-green light when the plate was heated. Thesolution was evaporated, redissolved in a small amount of CH₂ Cl₂ andfiltered. The product was separated by preparative TLC using 20% ethylacetate in hexane yielding 45 mg of the product as a slightly yellowoil: ¹ H NMR (CDCl₃) δ1.05 (m, 1H), 1.27 (m, 1H), 1.44-2.04 (m, 10H),2.23 (br s, 1H), 3.098 (br s, 1H), 3.711 (dd, 1H, J=11, 7.7 Hz), 3.866(dd, 1H, J=11, 4.4 Hz), 5.912 (dd, 1H, J=7.7, 4.4 Hz), 5.641 (br s, 1H),6.929 (dd, 1H), 6.98-7.42 (m, 3H).

Example 18 Synthesis of Compound 17

4-(3-Hydroxyphenyl)-4-(2,2,3,3,3-pentafluoropropoxy)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!. The vinyl sulfide ofExample 2(b) (0.112 g) was irradiated for 10 min in CH₂ Cl₂ containingmethylene blue with continuous oxygen bubbling at -78° C. using a 1000 WNa lamp. The solution was poured into a solution of mercuric acetate(100 mg, 0.84 eq.) in 5 mL of 2,2,3,3,3-pentafluoro-1-propanol andstored at -13° C. over night. TLC indicated the formation of two newproducts, both of which emitted light on heating. The solution wasevaporated, the blue residue washed with ether and the ether evaporatedto produce an oil. This oil was redissolved in a small amount of CH₂ Cl₂and the product isolated by preparative TLC using 30% ethyl acetate inhexane yielding 41.2 mg of dioxetane 17 as a colorless oil in additionto some of the acetoxy-substituted dioxetane (12); dioxetane 17: ¹ H NMR(CDCl₃) δ1.04 (m, 1H), 1.28 (m, 1H), 1.46-2.14 (m, 10H), 2.23 (br s,1H), 3.017 (br s, 1H), 3.658 (m, 1H), 4.006 (m, 1H), 5.13 (s, 1H), 6.93(dd, 1H), 6.98-7.28 (br s, 2H), 7.338 (t, 1H).

Example 19 Synthesis of Compound 18

4-(2,2,3,3,4,4,4-Heptafluorobutoxy)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!. The vinyl sulfide ofExample 2(b) (0.100 g) was irradiated for 10 min in 20 mL of CH₂ Cl₂containing 2 mg of methylene blue with continuous oxygen bubbling at-78° C. using a 1000 W Na lamp. The solution was poured into a solutionof mercuric acetate (100 mg, 0.95 eq.) in 5 mL of2,2,3,3,4,4,4-heptafluoro-1-butanol. After one hour, TLC indicated theformation of two new products, both of which emitted light on heating.The solution was evaporated, the blue residue washed with ether and theether evaporated to produce an oil. This oil was redissolved in a smallamount of CH₂ Cl₂ and the product isolated by preparative TLC using 30%ethyl acetate in hexane yielding 26.3 mg of dioxetane 18 as a colorlessoil in addition to some of the acetoxy-substituted dioxetane 12;dioxetane 18: ¹ H NMR (CDCl₃) δ1.05 (m, 1H), 1.28 (m, 1H), 1.45-1.96 (m,10H), 2.24 (br s, 1H), 3.029 (br s, 1H), 3.71 (m, 1H), 4.04 (m, 1H),5.332 (br s, 1H), 6.935 (dd, 1H), 6.98-7.52 (br s, 2H), 7.338 (t, 1H);¹⁹ F NMR (CDCl₃) δ-127.65, -120.58, -120.55, -120.52, -120.48, -120.46,-81.40, -81.36, -81.33.

Example 20 Synthesis of Compound 19

4-(2',6'-Difluorophenoxy)-4-(3-hydroxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!. The vinyl sulfide ofExample 2(b) (0.100 g) was irradiated for 10 min in 20 mL of CH₂ Cl₂containing 2 mg of methylene blue with continuous oxygen bubbling at-40° C. using a 1000 W Na lamp. NCS (44.7 mg, 1 eq.) was added followedafter 15 min by 100 mg (2.3 eq.) of 2,6-difluorophenol in 3 mL of CH₂Cl₂. The solution was allowed to warm to room temperature. After anhour, TLC showed a new material which emitted blue-green light when theplate was heated. The solution was evaporated, redissolved in a smallamount of CH₂ Cl₂ and the product isolated by two successive preparativeTLC purifications first using 5-20% ethyl acetate in hexane and thenusing CH₂ Cl₂ on a second plate yielding 11.2 mg of the product as aslightly yellow oil; ¹ H NMR (CDCl₃) δ1.29-2.24 (m, 13H), 3.31 (br s,1H), 5.00 (m, 1H), 6.68-7.6 (m, 7H).

Example 21 Synthesis of Compound 20

4-Methoxy-4-(3-pivaloyloxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo-3.3.1.1³,7 !decane!. The vinyl sulfide of Example 4(a) (0.051 g) wasirradiated in 20 mL of CH₃ OH containing ca. 1 mg of Rose Bengal withcontinuous oxygen bubbling at -78° C. using a 1000 W Na lamp. When thealkene was shown by TLC to be completely consumed, iodine (0.096 g) and1 mL of 30% H₂ O₂ were added and the mixture warmed to room temperature.Progress of the reaction was monitored by the disappearance of dioxetane4 by TLC (20% ethyl acetate in hexane) and the appearance of new bandswhich emitted blue light when the plate was heated and by observing thecolor of chemiluminescence from an aliquot of the reaction solutionreacted with 0.1 M TBAF in DMSO. The yellow emission of dioxetane 4 wasgradually replaced by the blue emission of dioxetane 20 over a period oftwo hours. The solution was concentrated to 1-2 mL and separated onpreparative TLC with 5% ethyl acetate in hexane. The band containingdioxetane 20 was collected and desorbed and evaporated to yield theproduct as a slightly yellow oil: ¹ H NMR (CDCl₃) δ1.02 (m, 1H), 1.28(m, 1H), 1.375 (s, 9H), 1.45-1.94 (m, 10H), 2.17 (br s, 1H), 3.04 (br s,1H), 3.232 (s, 3H), 7.07-7.49 (m, 4H).

Example 22 Synthesis of Compound 21

4-(3-t-Butyldimethylsilyloxyphenyl)-4-methoxyspiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. This exampledemonstrates the interconversion of triggerable dioxetanes by replacingone protecting group with another. A 152 mg portion (1 mmol) oft-butyldimethylsilyl chloride was added to a solution of imidazole (69mg, 1 mmol) in 2 mL of CH₂ Cl₂ causing formation of a white precipitate.The mixture was filtered and the supernatant transferred into a solutioncontaining 285 mg (0.94 mmol) of dioxetane 6 in 1 mL of CH₂ Cl₂. Afterstanding for 1 h, the solution was filtered, washed with 2×1 mL ofwater, dried and evaporated. The residue was dissolved in hexane,filtered and evaporated producing 310 mg of dioxetane 21 as an oil whichproduced blue chemiluminescence on heating or addition to a solution of0.1 M TBAF in DMSO. ¹ H NMR were identical to those reported for thiscompound in U.S. Pat. No. 4,857,652.

Example 23 Synthesis of Compound 22

4-(3-t-Butyldiphenylsilyloxyphenyl)-4-methoxyspiro1,2-dioxetane-3,2'-tricyclo- 3.3.1.1³,7 !decane!. This example furtherdemonstrates the interconversion of triggerable dioxetanes. A.950 mgportion (3.45 mmol) of t-butyldiphenylsilyl chloride was added to asolution of imidazole (236 mg, 3.47 mmol) in 25 mL of CH₂ Cl₂ causingformation of a white precipitate. The mixture was filtered and 0.99 g(3.3 mmol) of dioxetane 6 was added to the supernatant. After stirringover night, the solution was filtered, washed with 3×20 mL of water,dried and evaporated. The residue was dissolved in hexane, washed withwater, dried and allowed to crystallize at 4° C. Dioxetane 22 (767 mg)was obtained which produced blue chemiluminescence on heating oraddition to a solution of 0.1 M TBAF in DMSO. ¹ H NMR (CDCl₃) δ0.75-0.82(m, 1H), 1.10 (s, 9H), 1.35-1.87 (m, 11H), 2.03 (br s, 1H), 2.85 (br s,4H), 6.9-7.75 (m, 14H).

Example 24 Synthesis of Compound 23

4-(Methoxy)-4-(3-bis-(cyanoethyl)phosphoryloxyphenyl)spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!. Examples 24 and 25further demonstrate the interconversion of triggerable dioxetanes. Asolution of anhydrous pyridine (3.0 mL, 37 mmol) in 10 mL of CH₂ Cl₂ wasplaced under argon and cooled to 0° C. POCl₃ (1.608 g, 10.5 mmol) wasadded and the solution stirred for 15 min to cool. A solution ofdioxetane 6 (1.006 g, 3.3 mmol) and pyridine (3.0 mL) in 10 mL of CH₂Cl₂ was added dropwise. The ice bath was removed and the reactioncontinued as the solution warmed to room temperature. TLC (3:1 ethylacetate/hexane) showed complete conversion of dioxetane 6 in 90 min. Asolution of 2-cyanoethanol (2.2 mL, 32 mmol) and 3.0 mL of pyridine wasadded and stirring maintained over night. The solution was evaporated todryness yielding a white solid which was purified by chromatography onsilica with 30-75% ethyl acetate in hexane yielding dioxetane 23 as aslightly yellow oil (1.24 g): ¹ H NMR (CDCl₃) δ0.97 (d, 1H), 1.46-1.89(m, 11H), 2.103 (br s, 1H), 2.819 (t, 4H), 3.039 (br s, 1H), 3.224 (s,3H), 4.32-4.50 (m, 4H), 7.30-7.60 (m, 4H); ¹³ C NMR (CDCl₃) δ19.329,19.420, 25.521, 25.642, 31.197, 31.379, 31.925, 32.593, 32.836, 34.414,35.932, 49.773, 62.825, 62.916, 95.121, 111.057, 116.156, 120.648,120.709, 129.724, 136.948, 149.757, 149.848; ³¹ P NMR (CDCl₃) δ9.45 (m)rel to ext. H₃ PO₄.

Example 25 Synthesis of Compound 24

4-(Methoxy)-4-(3-phosphoryloxyphenyl)spiro 1,2-dioxetane-3,2'-tricyclo-3.3.1.1³,7 !decane!, disodium salt. To a solution of dioxetane 23 (1.24g, 2.54 mmol) dissolved in 50 mL of methanol was added a solution of1.627 g (15.35 mmol) of sodium carbonate in 10 mL of Type I water(Lumigen, Southfield, Mich.). After stirring for 2 days, TLC using 30%methanol in CH₂ Cl₂ showed complete removal of the cyanoethyl groups.The solid material was filtered off and washed with 50 mL of methanol.The methanol washes and reaction solution were combined and evaporatedunder reduced pressure yielding a white solid. The solid was freed ofimpurities by again dissolving in methanol, filtering and evaporatingthe methanol and crystallizing from methanol/acetone yielding 0.986 g ofwhite solid which was identical by ¹ H NMR to an authentic sample (seeU.S. Pat. No. 5,004,565).

Example 26 Alternate Synthesis of Vinyl Sulfide from Example 1(b) byTi-Mediated Coupling

(a) Synthesis of ethyl 3-methoxythiobenzoate. To an ice cooled solutionof m-anisoyl chloride (8.53 g) in 10 ml of CH₂ Cl₂ was added 4.85 mL ofpyridine. After 10 min, ethanethiol (3.73 g) was added dropwise and theresulting solution stirred over night. The solution was diluted with 100mL of methylene chloride, washed with saturated sodium bicarbonate thenwashed several times with water and dried. The thioester was obtained byevaporation of the solvent; ¹ H NMR (CDCl₃) δ1.35 (t, 3H), 3.06 (q, 2H),3.85 (s, 3H), 7.11-7.56 (m, 4H); ¹³ C NMR (CDCl₃) δ14.76, 23.53, 55.46,111.43, 119.69, 119.78, 129.58, 138.60, 159.76, 192.02.

(b) Synthesis of (3-methoxyphenyl)(ethylthio)methylene!tricyclo3.3.1.13,7!decane. A three neck flask was purged with argon and chargedwith 100 mL of anhydrous THF. The flask was cooled in an ice bath andtitanium trichloride (14.19 g) was added with stirring. Lithium aluminumhydride (LAH) (1.75 g) was added in small portions causing a briefexothermic reaction. After all of the LAH was added, the cooling bathwas removed and triethylamine (12.6 mL) was added. The black mixture wasrefluxed for 100 min under argon and then cooled for 10 min. A solutionof adamantanone (3.46 g) and ethyl 3-methoxythiobenzoate (1.5 g) in 50mL of dry THF was added dropwise over 20 min. Reaction progress wasmonitored by TLC with 20% ethyl acetate in hexane. The crude reactionmixture was cooled to room temperature after 120 min, diluted withhexane and decanted. The residue was washed several times using a totalof ca. 500 mL of hexane. The combined hexane solutions were filtered andevaporated leaving an oil. The alkene was purified by subjecting the oilto prep. TLC (15% ethyl acetate/hexane; ¹ H NMR (CDCl₃) δ1.09-1.14 (t,3H), 1.75-1.98 (m, 12H), 2.21-2.28 (q, 3H), 2.66 (s, 1H), 3.60 (s, 1H),3.82 (s, 3H), 6.78-7.23 (m, 4H); ¹³ C NMR (CDCl₃) δ15.21, 25.96, 28.17,28.48, 32.45, 35.45, 35.73, 37.07, 37.25, 39.10, 39.46, 39.98, 55.19,112.04, 115.01, 121.08, 122.06, 128.85, 141.57, 152.62, 159.36.

(c) Synthesis of (3-hydroxyphenyl)ethylthiomethylene!tricyclo-3.3.1.1³,7 !decane. A solution of the vinyl sulfide from step (b)dissolved in dry DMF is added to a solution of sodium ethanethiolate indry DMF under an atmosphere of argon. The mixture is refluxed for 3hours or until TLC indicates cleavage of the methyl ether. The mixtureis cooled to room temperature and carefully neutralized with diluteacid. The aqueous solution is extracted with ethyl acetate, the ethylacetate washed with water, dried and evaporated. The residual product ispurified by column chromatography.

Example 27 Comparison of Rates of Base-Induced Decay of HydroxyphenylAlkoxy or Aryloxy Dioxetanes

The first order decay of chemiluminescence of dioxetanes 14-19 in 0.2 M2-methyl-2-amino-1-propanol (221) buffer, pH 9.6 containing 0.88 mM Mg⁺²and 1.0 mg/mL of1-trioctylphosphoniummethyl-4-tributylphosphoniummethylbenzenedichloride was measured at 37° C. in a Turner Designs (Sunnyvale,Calif.) model TD-20e luminometer. The half-lives of decay ofchemiluminescence (t_(1/2)) of hydroxy-dioxetanes correlate with thetimes required to reach the maximum light intensity (I_(max)) in thealkaline phosphatase-triggered decomposition of the correspondingphosphate-dioxetanes under the same conditions. The half-life of decayof luminescence of the hydroxy dioxetane is, therefore, useful forpredicting the grow-in kinetics of light emission for phosphatasetriggering of phosphate dioxetanes; i.e. a fast t_(1/2) for the hydroxydioxetane indicates that the corresponding phosphate dioxetane isexpected to reach I_(max) more quickly. It is expected that, forexample, the phosphate derivatives of dioxetanes 14-19 as well as otherswhich can be produced by the process described herein will be useful inalkaline phosphatase-linked assays known in the art.

                  TABLE 3    ______________________________________    Kinetics of Light Emission from Hydroxy Dioxetanes    Prepared by the Process of the Present Invention.    Dioxetane    t1/2 (min) 37° C.    ______________________________________    14           0.16    15           3.7    16           4.1    17           3.4    18           4.9    19           1.76    ______________________________________

Example 28 Comparison of Rates of Base-Induced Decay of a Hydroxyphenylalkylthio Dioxetane

The first order decay of chemiluminescence of dioxetane 3 in 0.2 M 221buffer, pH 9.6 containing 0.88 mM Mg⁺² and the surfactant enhancersshown below was measured at ambient temperature. Surfactant A is CTAB, Bis poly(vinylbenzyltributylphosphonium chloride), C ispoly(vinylbenzyltributylphosphoniumchloride)-co-poly(vinylbenzyltrioctylphosphonium chloride), D is1-trioctylphoniummethyl-4-tributylphosphonium-methylbenzene dichloride.

The results indicate the surprising discovery that thesulfur-substituted dioxetane exhibits kinetics for light emissioncomparable to dioxetane 6 in buffer.

                  TABLE 4    ______________________________________    Kinetics of Light Emission from Dioxetane 3 in the    Presence of Surfactants.    Surfactant    Concentration                             t1/2 (min)    ______________________________________    A             0.41 g/L   11.2    B             0.5 g/L    4.2    C             0.5 g/L    2.9    D             1.0 g/L    2.0    ______________________________________

Example 29 Fluoride Induced Chemiluminescence of Alkoxy andAryloxy-Dioxetanes

A portion of each of the purified dioxetanes 6-23 was separately mixedwith a solution of 0.1 M tetrabutylammonium fluoride (TBAF) in DMSOcausing a brief flash of blue-green light which could be seen in adarkened room by eye. Chemiluminescence persisted for several seconds.Light emission produced in this manner could also be produced with thedioxetane deposited on a silica gel TLC plate.

Example 30 Fluoride Induced Chemiluminescence from Sulfur-SubstitutedDioxetanes

A portion of each of the dioxetanes 1-5 was separately mixed with asolution of 0.1 M TBAF in DMSO causing a brief flash of yellow toreddish light which could be seen in a darkened room by eye.Chemiluminescence persisted for several seconds.

We claim:
 1. A process for producing a stable triggerable dioxetanecomprising;(a) reacting a vinyl sulfide compound containing asulfur-substituent SR₄, wherein R₄ is an organic group containing 1 to20 carbon atoms and optionally heteroatoms, with oxygen and light in thepresence of a photosensitizer to form an intermediate sulfur-substituteddioxetane compound; and (b) reacting the sulfur-substituted dioxetanecompound with an electrophilic compound E--Y and a hydroxylic compoundR₅ OH selected from the group consisting of alcohols, phenols andcarboxylic acids or their salts and containing an OR₅ group to replacethe SR₄ group of the dioxetane with the OR₅ group wherein R₅ is selectedfrom the group consisting of (C₁ -C₂₀) alkyl groups, (C₂ -C₂₀) alkenylgroups, (C₂ -C₂₀) alkynyl groups, C₆ -C₃₀) aryl groups, (C₇ -C₃₀)aralkyl groups and (C₁ -C₂₀) acyl groups which can optionally besubstituted; E--Y is selected from the group consisting of halogens,hydrogen peroxide, singlet oxygen, pseudo-halogens, alkylating agents,transition metal salts, Lewis acids and (R₅ O⁻)_(n) M^(+n) where M is atransition metal; and n is 1, 2 or
 3. 2. The process of claim 1 whereinthe stable triggerable dioxetane is of the formula: ##STR33## wherein R₅is selected from the group consisting of (C₁ -C₂₀) alkyl groups, (C₂-C₂₀) alkenyl groups, (C₂ -C₂₀) alkynyl groups, (C₆ -C₃₀) aryl groups,(C₇ -C₃₀ ) aralkyl groups and (C₁ -C₂₀) acyl groups which can optionallybe substituted, wherein R₁ and R₂ are organic groups which providestability and which can optionally be joined together to form a cyclicor polycyclic group which is spiro-fused to the dioxetane ring and whichcan optionally be substituted, wherein R₃ is selected from aryl, biaryl,heteroaryl, fused ring polycyclic aryl and fused ring polycyclicheteroaryl groups which can optionally be substituted and wherein X is agroup which can be removed by an activating agent to form an unstableoxide intermediate dioxetane compound which decomposes and releaseselectronic energy to form light comprising;(a) reacting a vinyl sulfidecompound of the formula: ##STR34## containing a sulfur-substituent SR₄,wherein R₄ is an organic group containing 1 to 20 carbon atoms andoptionally heteroatoms, with oxygen and light in the presence of aphotosensitizer to form an intermediate sulfur-substituted dioxetanecompound of the formula: ##STR35## and (b) reacting thesulfur-substituted dioxetane compound with an electrophilic compoundE--Y and a hydroxylic compound R₅ OH selected from the group consistingof alcohols, phenols and carboxylic acids or their salts and containingan OR₅ group to replace the SR₄ group of the sulfur-substituteddioxetane with the OR₅ group.
 3. The process of claim 2 wherein thestable triggerable dioxetane has the formula: ##STR36## wherein##STR37## is a polycyclic organic group which is spiro-fused to thedioxetane ring and which can optionally be substituted comprising;(a)reacting a vinyl sulfide compound of the formula: ##STR38## containing asulfur-substituent SR₄, wherein R₄ is an organic group containing 1 to20 carbon atoms and optionally heteroatoms, with oxygen and light in thepresence of a photosensitizer to form an intermediate sulfur-substituteddioxetane compound of the formula: ##STR39## and (b) reacting thesulfur-substituted dioxetane compound with the electrophilic compoundE--Y and the hydroxylic compound to replace the SR₄ group of thesulfur-substituted dioxetane with the OR₅ group.
 4. The process of anyof claims 1, 2 or 3 wherein R₄ is selected from the group consisting ofalkyl containing 1 to 12 carbon atoms which can optionally besubstituted with at least one halogen atom and aryl containing 6 to 20carbon atoms which can optionally be substituted with at least onehalogen atom.
 5. The process of claim 4 wherein R₄ is a CH₂ CF₃ group.6. The process of claim 4 wherein R₄ is a CH₂ CH₃ group.
 7. The processof claim 4 wherein R₄ is a 4-fluorophenyl group.
 8. The process of claim3 wherein ##STR40## is an adamantyl group optionally substituted withnon-hydrogen groups.
 9. The process of claim 3 wherein R₃ is selectedfrom phenyl and naphthyl groups and wherein OX is selected from thegroup consisting of hydroxyl, alkoxy, substituted alkoxy, acyloxy havingthe formula OOCR₁₀ wherein R₁₀ is selected from alkyl and aryl groupscontaining 2 to 20 carbon atoms, trialklysilyloxy, triarylsilyloxy,aryldialkylsilyloxy, OPO(OR₈)₂ wherein R₈ is an organic group,β-D-galactosyloxy and β-D-glucuronidyloxy groups.
 10. The process ofclaim 8 wherein the sulfur-substituted dioxetane compound has theformula: ##STR41##
 11. The process of claim 10 wherein thesulfur-substituted dioxetane compound has the formula:
 12. The processof claim 10 wherein the sulfur-substituted dioxetane compound has theformula:
 13. The process of claim 10 wherein the sulfur-substituteddioxetane compound has the formula:
 14. The process of claim 10 whereinthe sulfur-substituted dioxetane compound has the formula:
 15. Theprocess of claim 1 wherein the electrophilic compound E--Y is selectedfrom the group consisting of halogens including Cl₂, Br₂, I₂, ICl andIBr, hydrogen peroxide, pseudo-halogens selected fromN-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide, mercurysalts, silver salts, gold salts, titanium tetrachloride and alkylatingagents selected from alkyl halides, alkyl sulfates and alkyl sulfonates.16. The process of claim 15 wherein the electrophilic compound E--Y isN-chlorosuccinimide.
 17. The process of claim 15 wherein theelectrophilic compound E--Y is mercuric acetate.
 18. The process ofclaim l wherein the vinyl sulfide is reacted with oxygen, light and thephotosensitizer at a temperature below about -30° C.
 19. The process ofclaim 1 wherein the hydroxylic compound is used as a solvent forreacting the vinyl sulfide to produce the sulfur-substituted dioxetane.20. The process of claim 1 wherein the electrophilic compound E--Y is ahydroxylic compound salt of the formula (R₅ O⁻)_(n) M^(+n) wherein M isa metal with a strong propensity to react with sulfur selected fromsilver, gold and mercury, wherein n is 1, 2 or 3 and wherein R₅ O⁻ is ananion of a hydroxylic compound.
 21. The process of claim 9 wherein theR₆ C group is an adamantyl group which is spiro-fused to the dioxetanering and the triggerable dioxetane has the formula:
 22. The process ofclaim 21 wherein R₃ is a meta-phenyl group and the stable triggerabledioxetane has the formula:
 23. A sulfur-substituted dioxetane compoundof the formula: containing a sulfur-substituent SR₄, wherein R₄ is anorganic group containing 1 to 20 carbon atoms and optionallyheteroatoms, wherein R₁ and R₂ are organic groups which providestability selected from the group consisting of straight chain, branchedchain or cyclic alkyl, substituted alkyl and heteroalkyl groups andwhich can optionally be joined together to form a cyclic or polycyclicgroup which is spiro-fused to the dioxetane ring and which canoptionally be substituted, wherein R₃ is selected from aryl, biaryl,heteroaryl, fused ring polycyclic aryl and fused ring polycyclicheteroaryl groups which can include additional substituents and whereinX is a protecting group which can be removed by an activating agent toform an unstable oxide intermediate dioxetane compound which decomposesand releases electronic energy to produce light and two carbonylcompounds of the formula: ##STR42##
 24. A sulfur-substituted dioxetanecompound of the formula: containing a sulfur-substituent SR₄, wherein R₄is an organic group containing 1 to 20 carbon atoms and optionallyheteroatoms, wherein ##STR43## is a polycyclic alkyl group which isspiro-fused to the dioxetane ring and which can optionally besubstituted, wherein R₃ is selected from aryl, biaryl, heteroaryl, fusedring polycyclic aryl and fused ring polycyclic heteroaryl groups whichcan include additional substituents and wherein X is a protecting groupwhich can be removed by an activating agent to form an unstable oxideintermediate dioxetane compound which decomposes and releases electronicenergy to produce light and two carbonyl compounds of the formula:##STR44##
 25. The compound of claim 24 wherein R₄ is selected from thegroup consisting of alkyl containing 1 to 12 carbon atoms which may besubstituted with at least one halogen atom and aryl containing 6 to 20carbon atoms which may be substituted with at least one halogen atom.26. The compound of claim 24 wherein R₃ is selected from the groupconsisting of substituted and unsubstituted adamantyl groups.
 27. Thecompound of claim 24 wherein R₃ is a meta-phenyl group and wherein OX isselected from the group consisting of hydroxyl, alkoxy, substitutedalkoxy, acyloxy having the formula OOCR₁₀ wherein R₁₀ is selected fromalkyl and aryl groups containing 2 to 20 carbon atoms, trialklysilyloxy,triarylsilyloxy, aryldialkylsilyloxy, OPO(OR₈)₂ wherein R₈ is an organicgroup, β-D-galactosyloxy and β-D-glucuronidyloxy groups.
 28. Thecompound of claim 24 having the formula:
 29. A compound having theformula:
 30. A compound having the formula:
 31. A compound having theformula:
 32. A compound having the formula:
 33. A compound having theformula: